Methods for Producing Polypeptidies in Protease-Deficient Mutants of Trichoderma

Abstract

The present invention relates to mutants of a parent Trichoderma strain, comprising a polynucleotide encoding a polypeptide and one or more (several) genes selected from the group consisting of a first subtilisin-like serine protease gene, a first aspartic protease gene, a trypsin-like serine protease gene, a second subtilisin-like serine protease gene, and a second aspartic pro-tease gene, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of a first subtilisin-like serine protease, a first aspartic protease, a trypsin-like serine protease, a second subtilisin-like serine protease, and a second aspartic protease, respectively, compared to the parent Trichoderma strain when cultivated under identical conditions. The present invention also relates to methods of producing a polypeptide in such mutants and methods for producing such mutants.

Description

REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing filed electronically by EFS, which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to methods of producing polypeptides in enzyme-deficient Trichoderma mutant strains, the enzyme-deficient Trichoderma mutant strains, and methods of obtaining the enzyme-deficient Trichoderma mutant strains.[0003]2. Description of the Related Art[0004]Trichoderma has been shown to be useful as a host cell for the recombinant production of polypeptides having biological activity (WO 96 / 00787, WO 97 / 26330). Trichoderma hosts with the desirable traits of increased protein expression and secretion may not necessarily have the most desirable characteristics for successful fermentation. The fermentation may not be optimal because of the production of biological substances, e.g., enzymes, detrimental to the production, recovery, or application of a par...

AI Technical Summary

Benefits of technology

[0060]An advantage of the present invention is elimination or reduction of one or more (several) enzyme activities, which may be detrimental to the production, downstream processing, e.g., recovery, and / or application of a particular polypeptide of interest

[0061]In the methods of the present invention, the parent Trichoderma strain may be any Trichoderma strain such as a wild-type Trichoderma strain or a mutant thereof. The parent Trichoderma strain may be Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride; or the alternative sexual form thereof, i.e., Hypocrea.

[0062]In another aspect, the parent Trichoderma strain is Trichoderma harzianum. In another aspect, the parent Trichoderma strain is Trichoderma koningii. In another aspect, the parent Trichoderma strain is Trichoderma longibrachiatum. In another aspect, the parent Trichoderma strain is Trichoderma reesei. In another aspect, the parent Trichoderma strain is Trichoderma viride.

[0063]In another aspect, the parent Trichoderma reesei strain is Trichoderma reesei RutC30. In another aspect, the parent Trichoderma reesei strain is Trichoderma reesei TV10. In another aspect, the parent Trichoderma reesei strain is a mutant of Trichoderma reesei. In another aspect, the parent Trichoderma reesei strain is a morphological mutant of Trichoderma reesei (see WO 97 / 26330).

[0064]The enzyme-deficient Trichoderma mutant strain may be constructed by reducing or eliminating expression of one or more (several) genes selected from the group consisting of a subtilisin-like serine protease gene, an aspartic protease gene, and a trypsin-like serine protease gene using methods well known in the art, such as insertions, disruptions, replacements, or deletions. A portion of the gene can be modified such as the coding region or a control sequence required for expression of the coding region. Such a control sequence of a gene may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the gene. For example, a promoter sequence may be inactivated resulting in no expression or a weaker promoter may be substituted for the native promoter sequence to reduce expression of the coding sequence. Other control sequences for possible modification include, but are not limited to, a leader, propeptide sequence, signal sequence, transcription terminator, and transcriptional activator.

[0065]The Trichoderma mutant strains may be constructed by gene deletion techniques to eliminate or reduce expression of a gene. Gene deletion techniques enable the partial or complete removal of the gene thereby eliminating their expression. In such methods, deletion of the gene(s) is accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5′ and 3′ regions flanking the gene.

Problems solved by technology

The fermentation may not be optimal because of the production of biological substances, e.g., enzymes, detrimental to the production, recovery, or application of a particular polypeptide of interest.

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